Bacillus subtilis is able to synthesize a lipopeptide biosurfactant (known as surfactin), which is one of the most effective biosurfactants available. In our previous study, B. subtilis ATCC 21332 was used to produce surfactant and it was found that the addition of iron at an appropriate amount significantly improved the biosurfactant production. However, it also showed that excess addition of iron led to a sharp decrease in pH of the culture and surfactin in the broth disappeared rapidly once the pH fell below 5.0. This study reveals that the disappearance of surfactin at the acidic pH was due to the precipitation of surfactin triggered by the accumulation of extracellular acidic metabolites. The acidic material was isolated and purified from the broth and was characterized by GC-MS and IR spectroscopy. The results of GC-MS and IR analysis show that the molecular weight and molecular structure of the acidic metabolite resembled those of phthalic anhydride.